Tongue FACS Notebook
Enter the directory of the maca folder on your drive and the name of the tissue you want to analyze.
tissue_of_interest = "Tongue"Load the requisite packages and some additional helper functions.
library(here)
library(useful)
library(Seurat)
library(dplyr)
library(Matrix)
library(ontologyIndex)
cell_ontology = get_ontology('https://raw.githubusercontent.com/obophenotype/cell-ontology/master/cl-basic.obo', extract_tags='everything')
validate_cell_ontology = function(cell_ontology_class){
in_cell_ontology = sapply(cell_ontology_class, function(x) is.element(x, cell_ontology$name))
if (!all(in_cell_ontology)) {
message = paste0('"', cell_ontology_class[!in_cell_ontology], '" is not in the cell ontology
')
stop(message)
}
}
convert_to_cell_ontology_id = function(cell_ontology_class){
return(sapply(cell_ontology_class, function(x) as.vector(cell_ontology$id[cell_ontology$name == x])[1]))
}
save_dir = here('00_data_ingest', 'tissue_robj')# read the metadata to get the plates we want
plate_metadata_filename = here('00_data_ingest', '00_facs_raw_data', 'metadata_FACS.csv')
plate_metadata <- read.csv(plate_metadata_filename, sep=",", header = TRUE)
colnames(plate_metadata)[1] <- "plate.barcode"
plate_metadataSubset the metadata on the tissue.
tissue_plates = filter(plate_metadata, tissue == tissue_of_interest)[,c('plate.barcode','tissue','subtissue','mouse.sex')]
tissue_platesLoad the read count data.
#Load the gene names and set the metadata columns by opening the first file
filename = here('00_data_ingest', '00_facs_raw_data', 'FACS', paste0(tissue_of_interest, '-counts.csv'))
raw.data = read.csv(filename, sep=",", row.names=1)
# raw.data = data.frame(row.names = rownames(raw.data))
corner(raw.data)Make a vector of plate barcodes for each cell
plate.barcodes = lapply(colnames(raw.data), function(x) strsplit(strsplit(x, "_")[[1]][1], '.', fixed=TRUE)[[1]][2])
head(plate.barcodes)[[1]]
[1] "D041894"
[[2]]
[1] "D041894"
[[3]]
[1] "D041894"
[[4]]
[1] "D041894"
[[5]]
[1] "D041894"
[[6]]
[1] "D041894"Use only the metadata rows corresponding to Bladder plates. Make a plate barcode dataframe to “expand” the per-plate metadata to be per-cell.
barcode.df = t.data.frame(as.data.frame(plate.barcodes))
rownames(barcode.df) = colnames(raw.data)
colnames(barcode.df) = c('plate.barcode')
head(barcode.df) plate.barcode
A15.D041894.3_9_M.1.1 "D041894"
B19.D041894.3_9_M.1.1 "D041894"
C21.D041894.3_9_M.1.1 "D041894"
E1.D041894.3_9_M.1.1 "D041894"
F4.D041894.3_9_M.1.1 "D041894"
G7.D041894.3_9_M.1.1 "D041894" rnames = row.names(barcode.df)
meta.data <- merge(barcode.df, plate_metadata, by='plate.barcode', sort = F)
row.names(meta.data) <- rnames
# Sort cells by plate barcode because that's how the data was originally
meta.data = meta.data[order(meta.data$plate.barcode), ]
corner(meta.data)raw.data = raw.data[, rownames(meta.data)]
corner(raw.data)Process the raw data and load it into the Seurat object.
# Find ERCC's, compute the percent ERCC, and drop them from the raw data.
erccs <- grep(pattern = "^ERCC-", x = rownames(x = raw.data), value = TRUE)
percent.ercc <- Matrix::colSums(raw.data[erccs, ])/Matrix::colSums(raw.data)
ercc.index <- grep(pattern = "^ERCC-", x = rownames(x = raw.data), value = FALSE)
raw.data <- raw.data[-ercc.index,]
# Create the Seurat object with all the data
tiss <- CreateSeuratObject(raw.data = raw.data, project = tissue_of_interest,
min.cells = 5, min.genes = 5)
tiss <- AddMetaData(object = tiss, meta.data)
tiss <- AddMetaData(object = tiss, percent.ercc, col.name = "percent.ercc")
# Change default name for sums of counts from nUMI to nReads
colnames(tiss@meta.data)[colnames(tiss@meta.data) == 'nUMI'] <- 'nReads'
# Create metadata columns for cell_ontology_classs and subcell_ontology_classs
tiss@meta.data[,'cell_ontology_class'] <- NA
tiss@meta.data[,'subcell_ontology_class'] <- NACalculate percent ribosomal genes.
ribo.genes <- grep(pattern = "^Rp[sl][[:digit:]]", x = rownames(x = tiss@data), value = TRUE)
percent.ribo <- Matrix::colSums(tiss@raw.data[ribo.genes, ])/Matrix::colSums(tiss@raw.data)
tiss <- AddMetaData(object = tiss, metadata = percent.ribo, col.name = "percent.ribo")A sanity check: genes per cell vs reads per cell.
GenePlot(object = tiss, gene1 = "nReads", gene2 = "nGene", use.raw=T)
Filter out cells with few reads and few genes.
tiss <- FilterCells(object = tiss, subset.names = c("nGene", "nReads"),
low.thresholds = c(500, 50000), high.thresholds = c(25000, 2000000))Normalize the data, then regress out correlation with total reads
tiss <- NormalizeData(object = tiss, scale.factor = 1e6)Performing log-normalization
0% 10 20 30 40 50 60 70 80 90 100%
|----|----|----|----|----|----|----|----|----|----|
**************************************************|tiss <- ScaleData(object = tiss)[1] "Scaling data matrix"
|
| | 0%
|
|====================================================================| 100%tiss <- FindVariableGenes(object = tiss, do.plot = TRUE, x.high.cutoff = Inf, y.cutoff = 0.5)Calculating gene means
0% 10 20 30 40 50 60 70 80 90 100%
|----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating gene variance to mean ratios
0% 10 20 30 40 50 60 70 80 90 100%
|----|----|----|----|----|----|----|----|----|----|
**************************************************|
Run Principal Component Analysis.
tiss <- RunPCA(object = tiss, do.print = FALSE)
tiss <- ProjectPCA(object = tiss, do.print = FALSE)
Later on (in FindClusters and TSNE) you will pick a number of principal components to use. This has the effect of keeping the major directions of variation in the data and, ideally, supressing noise. There is no correct answer to the number to use, but a decent rule of thumb is to go until the plot plateaus.
PCElbowPlot(object = tiss)
Choose the number of principal components to use.
# Set number of principal components.
n.pcs = 10The clustering is performed based on a nearest neighbors graph. Cells that have similar expression will be joined together. The Louvain algorithm looks for groups of cells with high modularity–more connections within the group than between groups. The resolution parameter determines the scale…higher resolution will give more clusters, lower resolution will give fewer.
For the top-level clustering, aim to under-cluster instead of over-cluster. It will be easy to subset groups and further analyze them below.
# Set resolution
res.used <- 0.5
tiss <- FindClusters(object = tiss, reduction.type = "pca", dims.use = 1:n.pcs,
resolution = res.used, print.output = 0, save.SNN = TRUE, force.recalc = TRUE)To visualize
# If cells are too spread out, you can raise the perplexity. If you have few cells, try a lower perplexity (but never less than 10).
tiss <- RunTSNE(object = tiss, dims.use = 1:n.pcs, seed.use = 10, perplexity=30)
# note that you can set do.label=T to help label individual clusters
TSNEPlot(object = tiss, do.label = T, pt.size = 1.2, label.size = 4)
Check expression of genes of interset.
Dotplots let you see the intensity of exppression and the fraction of cells expressing for each of your genes of interest.
How big are the clusters?
table(tiss@ident)
0 1 2 3 4 5
461 274 194 178 163 124 tiss1=BuildClusterTree(tiss)[1] "Finished averaging RNA for cluster 0"
[1] "Finished averaging RNA for cluster 1"
[1] "Finished averaging RNA for cluster 2"
[1] "Finished averaging RNA for cluster 3"
[1] "Finished averaging RNA for cluster 4"
[1] "Finished averaging RNA for cluster 5"
marker10=FindAllMarkersNode(tiss1, 10)
| | 0 % ~calculating
|+ | 1 % ~20s
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|++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed = 19s
| | 0 % ~calculating
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|++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed = 10sprint(x = head(x= marker10, n = 30))Which markers identify a specific cluster?
clust.markers <- FindMarkers(object = tiss, ident.1 = 2, only.pos = TRUE, min.pct = 0.25, thresh.use = 0.25)
| | 0 % ~calculating
|+ | 1 % ~26s
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|++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed = 28sprint(x = head(x= clust.markers, n = 30))You can also compute all markers for all clusters at once. This may take some time.
tiss.markers <- FindAllMarkers(object = tiss, only.pos = TRUE, min.pct = 0.25, thresh.use = 0.25)
| | 0 % ~calculating
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|+++++++++++++++++++++++++++ | 53% ~28s
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|+++++++++++++++++++++++++++++++ | 61% ~23s
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|+++++++++++++++++++++++++++++++++ | 64% ~21s
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|+++++++++++++++++++++++++++++++++++++ | 73% ~16s
|++++++++++++++++++++++++++++++++++++++ | 74% ~15s
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|+++++++++++++++++++++++++++++++++++++++ | 77% ~14s
|+++++++++++++++++++++++++++++++++++++++ | 78% ~13s
|++++++++++++++++++++++++++++++++++++++++ | 79% ~13s
|++++++++++++++++++++++++++++++++++++++++ | 80% ~12s
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|+++++++++++++++++++++++++++++++++++++++++ | 82% ~11s
|++++++++++++++++++++++++++++++++++++++++++ | 83% ~10s
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|+++++++++++++++++++++++++++++++++++++++++++ | 85% ~09s
|+++++++++++++++++++++++++++++++++++++++++++ | 86% ~09s
|++++++++++++++++++++++++++++++++++++++++++++ | 87% ~08s
|++++++++++++++++++++++++++++++++++++++++++++ | 88% ~07s
|+++++++++++++++++++++++++++++++++++++++++++++ | 89% ~07s
|+++++++++++++++++++++++++++++++++++++++++++++ | 90% ~06s
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|+++++++++++++++++++++++++++++++++++++++++++++++ | 93% ~04s
|+++++++++++++++++++++++++++++++++++++++++++++++ | 94% ~04s
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|+++++++++++++++++++++++++++++++++++++++++++++++++ | 98% ~01s
|++++++++++++++++++++++++++++++++++++++++++++++++++| 99% ~01s
|++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed = 59s
| | 0 % ~calculating
|+ | 1 % ~01m 33s
|++ | 2 % ~01m 29s
|++ | 3 % ~01m 27s
|+++ | 4 % ~01m 26s
|+++ | 5 % ~01m 26s
|++++ | 6 % ~01m 24s
|++++ | 7 % ~01m 23s
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|+++++ | 9 % ~01m 21s
|++++++ | 10% ~01m 20s
|++++++ | 11% ~01m 19s
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|+++++++ | 13% ~01m 21s
|++++++++ | 14% ~01m 21s
|++++++++ | 15% ~01m 21s
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|++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed = 01m 56sDisplay the top markers you computed above.
tiss.markers %>% group_by(cluster)Assigning cell type identity to clusters
At a coarse level, we can use canonical markers to match the unbiased clustering to known cell types:
0: Basal/differentiating cells 1: Basal/differentiating cells 2: Proliferating cells 3: Maturing/nonkeratinizing cells 4: Basal/differentiating cells 5: Stratified/differentiated keratinocytes 6: Basal/differentiating cells 7: Filliform Keratinocytes
# stash current cluster IDs
tiss <- StashIdent(object = tiss, save.name = "cluster.ids")
# enumerate current cluster IDs and the labels for them
cluster.ids <- c(0, 1, 2, 3, 4, 5, 6, 7)
cell_ontology_class <- c("basal cell of epidermis", "basal cell of epidermis", "keratinocyte", "keratinocyte", "basal cell of epidermis", "keratinocyte", "basal cell of epidermis", "keratinocyte" )
cell_ontology_id <- c("CL:0000312", "CL:0000312", "CL:0000312", "CL:0000312", "CL:0000312", "CL:0000312", "CL:0000312", "CL:0000312" )
tiss@meta.data[,'cell_ontology_class'] <- plyr::mapvalues(x = tiss@ident, from = cluster.ids, to = cell_ontology_class)
tiss@meta.data[,'cell_ontology_id'] <- plyr::mapvalues(x = tiss@ident, from = cluster.ids, to = cell_ontology_id)
tiss@meta.data[tiss@cell.names,'cell_ontology_class'] <- as.character(tiss@meta.data$cell_ontology_class)
tiss@meta.data[tiss@cell.names,'cell_ontology_id'] <- as.character(tiss@meta.data$cell_ontology_id)
TSNEPlot(object = tiss, do.label = TRUE, pt.size = 1, label.size = 3, group.by='cell_ontology_class', do.return = TRUE)
#dev.copy(png,'annotated_plot-2.png')
#dev.off()Checking for batch effects
Color by metadata, like plate barcode, to check for batch effects.
TSNEPlot(object = tiss, do.return = TRUE, group.by = "mouse.sex")Print a table showing the count of cells in each identity category from each plate.
table(as.character(tiss@ident), as.character(tiss@meta.data$plate.barcode))Save the Robject for later
When you save the annotated tissue, please give it a name.
filename = here('00_data_ingest', '04_tissue_robj_generated',
paste0("facs", tissue_of_interest, "_seurat_tiss.Robj"))
print(filename)
save(tiss, file=filename)# To reload a saved object
filename = here('00_data_ingest', '04_tissue_robj_generated',
paste0("facs", tissue_of_interest, "_seurat_tiss.Robj"))
load(file=filename)Export the final metadata
So that Biohub can easily combine all your cell_ontology_classs, please export them as a simple csv.
head(tiss@meta.data)filename = here('00_data_ingest', '03_tissue_cell_ontology_class_csv',
paste0(tissue_of_interest, "_cell_ontology_class.csv"))
write.csv(tiss@meta.data[,c('plate.barcode','cell_ontology_class','cell_ontology_id')], file=filename)---
title: "Tongue FACS Notebook"
output: html_notebook
---

Enter the directory of the maca folder on your drive and the name of the tissue you want to analyze.

```{r}
tissue_of_interest = "Tongue"
```

Load the requisite packages and some additional helper functions.

```{r}
library(here)
library(useful)
library(Seurat)
library(dplyr)
library(Matrix)
library(ontologyIndex)
cell_ontology = get_ontology('https://raw.githubusercontent.com/obophenotype/cell-ontology/master/cl-basic.obo', extract_tags='everything')

validate_cell_ontology = function(cell_ontology_class){
  in_cell_ontology = sapply(cell_ontology_class, function(x) is.element(x, cell_ontology$name))
  if (!all(in_cell_ontology)) {
    message = paste0('"', cell_ontology_class[!in_cell_ontology], '" is not in the cell ontology
')
    stop(message)
  }
}
convert_to_cell_ontology_id = function(cell_ontology_class){
  return(sapply(cell_ontology_class, function(x) as.vector(cell_ontology$id[cell_ontology$name == x])[1]))
}

save_dir = here('00_data_ingest', 'tissue_robj')
```



```{r}
# read the metadata to get the plates we want
plate_metadata_filename = here('00_data_ingest', '00_facs_raw_data', 'metadata_FACS.csv')

plate_metadata <- read.csv(plate_metadata_filename, sep=",", header = TRUE)
colnames(plate_metadata)[1] <- "plate.barcode"
plate_metadata
```

Subset the metadata on the tissue.

```{r}
tissue_plates = filter(plate_metadata, tissue == tissue_of_interest)[,c('plate.barcode','tissue','subtissue','mouse.sex')]
tissue_plates
```

Load the read count data.
```{r}
#Load the gene names and set the metadata columns by opening the first file
filename = here('00_data_ingest', '00_facs_raw_data', 'FACS', paste0(tissue_of_interest, '-counts.csv'))

raw.data = read.csv(filename, sep=",", row.names=1)
# raw.data = data.frame(row.names = rownames(raw.data))
corner(raw.data)
```
Make a vector of plate barcodes for each cell

```{r}
plate.barcodes = lapply(colnames(raw.data), function(x) strsplit(strsplit(x, "_")[[1]][1], '.', fixed=TRUE)[[1]][2])
head(plate.barcodes)
```

Use only the metadata rows corresponding to Bladder plates. Make a plate barcode dataframe to "expand" the per-plate metadata to be per-cell.
```{r}
barcode.df = t.data.frame(as.data.frame(plate.barcodes))

rownames(barcode.df) = colnames(raw.data)
colnames(barcode.df) = c('plate.barcode')
head(barcode.df)

rnames = row.names(barcode.df)
meta.data <- merge(barcode.df, plate_metadata, by='plate.barcode', sort = F)
row.names(meta.data) <- rnames

# Sort cells by plate barcode because that's how the data was originally
meta.data = meta.data[order(meta.data$plate.barcode), ]
corner(meta.data)
raw.data = raw.data[, rownames(meta.data)]
corner(raw.data)
```
Process the raw data and load it into the Seurat object.

```{r}
# Find ERCC's, compute the percent ERCC, and drop them from the raw data.
erccs <- grep(pattern = "^ERCC-", x = rownames(x = raw.data), value = TRUE)
percent.ercc <- Matrix::colSums(raw.data[erccs, ])/Matrix::colSums(raw.data)
ercc.index <- grep(pattern = "^ERCC-", x = rownames(x = raw.data), value = FALSE)
raw.data <- raw.data[-ercc.index,]

# Create the Seurat object with all the data
tiss <- CreateSeuratObject(raw.data = raw.data, project = tissue_of_interest, 
                    min.cells = 5, min.genes = 5)

tiss <- AddMetaData(object = tiss, meta.data)
tiss <- AddMetaData(object = tiss, percent.ercc, col.name = "percent.ercc")
# Change default name for sums of counts from nUMI to nReads
colnames(tiss@meta.data)[colnames(tiss@meta.data) == 'nUMI'] <- 'nReads'

# Create metadata columns for cell_ontology_classs and subcell_ontology_classs
tiss@meta.data[,'cell_ontology_class'] <- NA
tiss@meta.data[,'subcell_ontology_class'] <- NA
```


Calculate percent ribosomal genes.

```{r}
ribo.genes <- grep(pattern = "^Rp[sl][[:digit:]]", x = rownames(x = tiss@data), value = TRUE)
percent.ribo <- Matrix::colSums(tiss@raw.data[ribo.genes, ])/Matrix::colSums(tiss@raw.data)
tiss <- AddMetaData(object = tiss, metadata = percent.ribo, col.name = "percent.ribo")
```

A sanity check: genes per cell vs reads per cell.

```{r}
GenePlot(object = tiss, gene1 = "nReads", gene2 = "nGene", use.raw=T)
```

Filter out cells with few reads and few genes.

```{r}
tiss <- FilterCells(object = tiss, subset.names = c("nGene", "nReads"), 
    low.thresholds = c(500, 50000), high.thresholds = c(25000, 2000000))
```


Normalize the data, then regress out correlation with total reads
```{r}
tiss <- NormalizeData(object = tiss, scale.factor = 1e6)
tiss <- ScaleData(object = tiss)
tiss <- FindVariableGenes(object = tiss, do.plot = TRUE, x.high.cutoff = Inf, y.cutoff = 0.5)
```


Run Principal Component Analysis.
```{r}
tiss <- RunPCA(object = tiss, do.print = FALSE)
tiss <- ProjectPCA(object = tiss, do.print = FALSE)
```

```{r, echo=FALSE, fig.height=4, fig.width=8}
PCHeatmap(object = tiss, pc.use = 1:3, cells.use = 1000, do.balanced = TRUE, label.columns = FALSE, num.genes = 8)
```

Later on (in FindClusters and TSNE) you will pick a number of principal components to use. This has the effect of keeping the major directions of variation in the data and, ideally, supressing noise. There is no correct answer to the number to use, but a decent rule of thumb is to go until the plot plateaus.

```{r}
PCElbowPlot(object = tiss)
```

Choose the number of principal components to use.
```{r}
# Set number of principal components. 
n.pcs = 10
```


The clustering is performed based on a nearest neighbors graph. Cells that have similar expression will be joined together. The Louvain algorithm looks for groups of cells with high modularity--more connections within the group than between groups. The resolution parameter determines the scale...higher resolution will give more clusters, lower resolution will give fewer.

For the top-level clustering, aim to under-cluster instead of over-cluster. It will be easy to subset groups and further analyze them below.

```{r}
# Set resolution 
res.used <- 0.5

tiss <- FindClusters(object = tiss, reduction.type = "pca", dims.use = 1:n.pcs, 
    resolution = res.used, print.output = 0, save.SNN = TRUE, force.recalc = TRUE)
```

To visualize 
```{r}
# If cells are too spread out, you can raise the perplexity. If you have few cells, try a lower perplexity (but never less than 10).
tiss <- RunTSNE(object = tiss, dims.use = 1:n.pcs, seed.use = 10, perplexity=30)
# note that you can set do.label=T to help label individual clusters
TSNEPlot(object = tiss, do.label = T, pt.size = 1.2, label.size = 4)

```


Check expression of genes of interset.

```{r, echo=FALSE, fig.height=12, fig.width=8}
genes_to_check = c('Top2a', 'Cdc20', 'Mki67', 'Krt5', 'Krt14', 'Krt15', 'Krt84', 'Krt36', 'Hoxc13', 'Krt4', 'Krt13', 'Krt78', 'Krt10', 'Sbsn', 'Dcn')

FeaturePlot(tiss, genes_to_check, pt.size = 1.5, nCol = 3)
```

Dotplots let you see the intensity of exppression and the fraction of cells expressing for each of your genes of interest.

```{r, echo=FALSE, fig.height=4, fig.width=8}
# To change the y-axis to show raw counts, add use.raw = T.
DotPlot(tiss, genes_to_check, plot.legend = T)
```

How big are the clusters?
```{r}
table(tiss@ident)
```

```{r}
tiss1=BuildClusterTree(tiss)
```

```{r}
marker10=FindAllMarkersNode(tiss1, 10)
```
```{r}
print(x = head(x= marker10, n = 30))
```



Which markers identify a specific cluster?

```{r}
clust.markers <- FindMarkers(object = tiss, ident.1 = 2, only.pos = TRUE, min.pct = 0.25, thresh.use = 0.25)
```

```{r}
print(x = head(x= clust.markers, n = 30))
```

You can also compute all markers for all clusters at once. This may take some time.
```{r}
tiss.markers <- FindAllMarkers(object = tiss, only.pos = TRUE, min.pct = 0.25, thresh.use = 0.25)
```

Display the top markers you computed above.
```{r}
tiss.markers %>% group_by(cluster)
```

## Assigning cell type identity to clusters

At a coarse level, we can use canonical markers to match the unbiased clustering to known cell types:

0: Basal/differentiating cells
1: Basal/differentiating cells
2: Proliferating cells
3: Maturing/nonkeratinizing cells
4: Basal/differentiating cells
5: Stratified/differentiated keratinocytes
6: Basal/differentiating cells
7: Filliform Keratinocytes

```{r}
# stash current cluster IDs
tiss <- StashIdent(object = tiss, save.name = "cluster.ids")

# enumerate current cluster IDs and the labels for them
cluster.ids <- c(0, 1, 2, 3, 4, 5, 6, 7)
cell_ontology_class <- c("basal cell of epidermis", "basal cell of epidermis", "keratinocyte", "keratinocyte", "basal cell of epidermis", "keratinocyte", "basal cell of epidermis", "keratinocyte" )
cell_ontology_id <- c("CL:0000312", "CL:0000312", "CL:0000312", "CL:0000312", "CL:0000312", "CL:0000312", "CL:0000312", "CL:0000312" )

tiss@meta.data[,'cell_ontology_class'] <- plyr::mapvalues(x = tiss@ident, from = cluster.ids, to = cell_ontology_class)
tiss@meta.data[,'cell_ontology_id'] <- plyr::mapvalues(x = tiss@ident, from = cluster.ids, to = cell_ontology_id)

tiss@meta.data[tiss@cell.names,'cell_ontology_class'] <- as.character(tiss@meta.data$cell_ontology_class)
tiss@meta.data[tiss@cell.names,'cell_ontology_id'] <- as.character(tiss@meta.data$cell_ontology_id)

TSNEPlot(object = tiss, do.label = TRUE, pt.size = 1, label.size = 3, group.by='cell_ontology_class', do.return = TRUE)
#dev.copy(png,'annotated_plot-2.png')
#dev.off()
```


## Checking for batch effects


Color by metadata, like plate barcode, to check for batch effects.
```{r}
TSNEPlot(object = tiss, do.return = TRUE, group.by = "mouse.sex")
```

Print a table showing the count of cells in each identity category from each plate.

```{r}
table(as.character(tiss@ident), as.character(tiss@meta.data$plate.barcode))
```



# Save the Robject for later
When you save the annotated tissue, please give it a name.

```{r}
filename = here('00_data_ingest', '04_tissue_robj_generated', 
                     paste0("facs", tissue_of_interest, "_seurat_tiss.Robj"))
print(filename)
save(tiss, file=filename)
```

```{r}
# To reload a saved object
filename = here('00_data_ingest', '04_tissue_robj_generated', 
                      paste0("facs", tissue_of_interest, "_seurat_tiss.Robj"))
load(file=filename)
```



# Export the final metadata

So that Biohub can easily combine all your cell_ontology_classs, please export them as a simple csv.

```{r}
head(tiss@meta.data)
```


```{r}
filename = here('00_data_ingest', '03_tissue_cell_ontology_class_csv', 
                     paste0(tissue_of_interest, "_cell_ontology_class.csv"))
write.csv(tiss@meta.data[,c('plate.barcode','cell_ontology_class','cell_ontology_id')], file=filename)
```


